Methods of altering bone growth by administration of Sost or Wise antagonist or agonist

ABSTRACT

The present invention provides a method of promoting local bone growth by administering a therapeutic amount of a Sost antagonist to a mammalian patient in need thereof. Preferably, the Sost antagonist is an antibody or FAB fragment selectively recognizing any one of SEQ ID NOS: 1-23. The Sost antagonist may be coadministered together or sequentially with a matrix conducive to anchoring new bone growth. Orthopedic and Periodontal devices comprising an implantable portion adapted to be permanently implanted within a mammalian body and bearing an external coating of a Sost antagonist are also disclosed, as it a method of increasing bone density by administering to a mammalian patient a therapeutic amount of a Sost antagonist together with an antiresorptive drug.

This application is a continuation of application Ser. No. 14/504,544filed Oct. 2, 2014 (now abandoned), which in turn is a continuation ofapplication Ser. No. 13/796,530 filed Mar. 12, 2013 (now U.S. Pat. No.8,877,196 issued Nov. 4, 2014), which in turn is a divisional of Ser.No. 13/420,846 filed Mar. 15, 2012, which in turn is a continuation ofapplication Ser. No. 11/962,522 filed Dec. 21, 2007 (now U.S. Pat. No.8,178,099 issued May 15, 2012), which in turn is a non provisional ofapplication No. 60/882,642 filed Dec. 29, 2006.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods of altering bone growth. Inparticular, the present invention relates to promoting local bone growthby administering therapeutic amounts of a Sclerostin (hereinaftersometimes “Sost”) or Wise antagonist with or without an osteoconductivescaffold to a mammal. In a different embodiment, the present inventionrelates to implantable medical devices comprising Sost or Wiseantagonists or agonists. In a different embodiment, the presentinvention relates to promoting new bone by systemic administration of aSost or Wise antagonist in combination with an antiresorptive agent. Inanother embodiment, the present invention relates to methods of reducingbone both systemically and locally by administering a therapeutic amountof a Sost or Wise agonist to a mammal. In a still further embodiment,the present invention relates to a method of protecting a mammaliankidney from any chemical injury or glomuleronephritis by administering aWise or Sost antagonist.

2. Brief Description of the Background Art

It is well-understood that bone formation is indicated for treatment ofa wide variety of disparate disorders in mammals including simple aging,bone degeneration and osteoporosis, fracture healing, fusion orarthrodesis, osteogenesis imperfecta, etc., as well as for successfulinstallation of various medical orthopedic and periodontal implants suchas screws, rods, titanium cage for spinal fusion, hip joints, kneejoint, ankle joints, shoulder joints, dental plates and rods, etc.Contrarywise, it is also understood that more rarely disorders appear inmammals wherein bone is overproduced such as in: heterotopicossification or osteosarcoma treatment, to prevent progression or reducespinal stenosis of osseous origin such as osteophyte or ossification ofthe posterior longitudinal ligament, to prevent spontaneous fusion ororthrodesis with joint or disc arthroplasty, to prevent or treatspontaneous spinal fusion such as diffuse idiopathic skeletalhyperostosis and ankylosing spondylitis, preventing ossification orcalcification of ligaments, tendons or joint capsules, treatingheterotopic bone formation, preventing systemic hyperostosis resultingfrom metabolic bone disease, and Paget's disease. For these indicationsand others it is desired to reduce or inhibit such overproduction whenpossible.

Increasing bone mineralization to treat conditions characterized atleast in part by increased bone resorption, such as osteopenia, bonefractures, osteoporosis, arthritis, tumor metastases, Paget's diseaseand other metabolic bone disorders, using cathepsin K inhibitors andTGF-beta binding proteins, etc., are well-known as shown by US patentapplication No. 20040235728 to Selwyn Aubrey Stoch, published Nov. 25,2004, and Mary E. Brunkow et al U.S. Pat. No. 6,489,445 and US patentapplication publication 20040009535, published Jan. 15, 2004. In theBrunkow '535 and '445 publication, the TGF-beta binding proteins includeSost polypeptide (full length and short peptide) antibodies thatinterfere with the interaction between the TGF-beta binding proteinsclerostin and a TGF-beta superfamily member, particularly a bonemorphogenic protein. All of the diseases named above are due to asystemic loss of bone mineral and thus the administration of theantibody therapeutic is for systemic (whole body) increase in bonemineral density.

In the Brunkow '445 and '535 patent, the binding proteins preferablybind specifically to at least one human bone morphogenic protein (BMP)among BMP-5 and BMP-6.

U.S. Pat. No. 6,395,511 to Brunkow, et al. teaches a novel family ofhuman TGF-beta binding proteins and nucleic acids encoding them. Theprotein binds to at least human bone morphogenic protein-5 and humanbone morphogenic protein-6.

Sclerosteosis is a progressive sclerosing bone dysplasia. Sclerostin(the Sost gene) was originally identified as the sclerosteosis-causinggene. Sclerostin was intensely expressed in developing bones of mouseembryos. Punctuated expression of sclerostin was localized on thesurfaces of both intramembranously forming skull bones andendochondrally forming long bones. The physiological role of sclerostinremains to be elucidated. However, it is known that loss of functionmutations in Sost cause a rare bone dysplasia characterized by skeletalovergrowth.

In-San Kim's US patent application No. 20060165799, published Jul. 27,2006, teaches a bone-filling composition for stimulating bone-formingand bone-consolidation comprising biocompatible calcium sulfate andviscous biopolymers. The composition is intended to be administeredeasily into the missing part of injured bone without diffusing tosurrounding organs.

In Ronald S. Sapieszko's U.S. Pat. No. 5,939,039, issued in 1999 teachesthe processes to yield unique calcium phosphate precursor minerals thatcan be used to form a self-setting cement or paste. Once placed in thebody, these calcium phosphate cements (CPC) will be resorbed andremodeled (converted) to bone.

For example, calcium phosphate particles prepared in accordance with the'039 patent can be used in any of the orthopaedic or dental proceduresknown for the use of calcium phosphate; the procedures of bone fillingdefect repair, oncological defect filling, craniomaxillofacial voidfilling and reconstruction, dental extraction site filling.

US patent application No. 20060198863 to Carl Alexander DePaula,published Sep. 7, 2006, relates to a formable ceramic composition forfilling bone defects. The composition comprises ceramic beta tricalciumphosphate particles having a particle size from about 40 microns to 500microns admixed with a hydrogel carrier containing citric acid buffer.The composition has a pH between 7.0 to 7.8 and the hydrogel componentof the carrier ranges from about 1.0 to 5.0% of the composition.

Wise and SOST are understood to be closely related family members(Ellies et al, JBMR 2006 November; 21(11):1738-49.). Those of ordinaryskill are aware that the Wise null mutant mouse exhibits a bonephenotype (Keynote presentation at the 2005 American Society of BoneMineral Research meeting in Nashville, Tenn. State of the Art lectures,an embryonic source of skeletal tissue. Patterning CraniofacialDevelopment; by Robb Krumlauf, Ph.D., Stowers Institute for MedicalResearch, Kansas City, Mo., USA

US patent application No. 2005025604 to Vignery published Nov. 17, 2005shows induction of bone formation by mechanically inducing an increasein osteoblast activity and elevating systemic blood concentration of abone anabolic agent, including optionally elevating systemic bloodconcentration of an antiresorptive agent.

Finally, Yanagita, Modulator of bone morphogenic protein activity in theprogression of kidney diseases, Kidney Int., Vol. 70, No. 6 (2006)989-93 shows Usag-1 (also known as “Wise”) protects the kidney fromcisplatin insult due to BMB inhibition. See also Yanagita, Uterinesensitization-associated gene-1 (USAG-1), a novel antagonist expressedin the kidney, accelerates tubular injury, J. Clin. Invest., Vol. 116,No. 1 (2005) 70-9, Yanagita, BMP antagonists: their roles in developmentand involvement in pathophysiology, Cytokine Growth Factor Rev., Vol 16,No. 3 (2005) 309-17, and Yanagita, USAG-1: a bone morphogenic proteinantagonist abundantly expressed in the kidney, Biochem. Biophys. Res.Commun., Vol. 316, No. 2 (2004) 490-500 and

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method ofpromoting local bone growth, comprising the steps of administeringlocally a therapeutic amount of a Sost antagonist to a mammalian patientin need thereof.

It is an object of the present invention to provide a method ofpromoting local bone growth, comprising the steps of administeringlocally a therapeutic amount of a Wise antagonist to a mammalian patientin need thereof.

It is an object of the present invention to provide a method ofpromoting local bone growth, comprising the steps of administeringlocally a therapeutic amount of a Sost antagonist in conjunction with anosteoconductive biocompatible calcium salt scaffold to a mammalianpatient in need thereof.

It is an object of the present invention to provide a method ofpromoting local bone growth, comprising the steps of administeringlocally a therapeutic amount of a Wise antagonist in conjunction with anosteoconductive biocompatible calcium salt scaffold to a mammalianpatient in need thereof.

It is a further object of the present invention to provide a medicalorthopedic or periodontal device, comprising a structural support,wherein an implantable portion of said structural support is adapted tobe permanently implanted within a mammalian body, said implantedstructure support being at least partially retained in said body bylocal bone growth, said structural support bearing an at least a partialexternal coating of a Sost antagonist with or without an osteoconductivebiocompatible scaffold.

It is a further object of the present invention to provide a medicalorthopedic or periodontal device, comprising a structural support,wherein an implantable portion of said structural support is adapted tobe permanently implanted within a mammalian body, said implantedstructure support being at least partially retained in said body bylocal bone growth, said structural support bearing an at least a partialexternal coating of a Wise antagonist with or without an osteoconductivebiocompatible scaffold.

It is a still further object of the present invention to provide amethod of increasing bone density both systemically (whole body) andlocally, comprising the steps of administering, to a mammalian patientin need thereof, a therapeutic amount of a Sost antagonist together withan antiresorptive drug.

It is a still further object of the present invention to provide amethod of increasing bone density both systemically (whole body) andlocally, comprising the steps of administering, to a mammalian patientin need thereof, a therapeutic amount of a Wise antagonist together withan antiresorptive drug.

It is a still further object of the present invention to provide amethod of reducing bone both locally and systemically (whole body),comprising the steps of administering a therapeutic amount of a Sostagonist to a mammalian patient in need thereof.

It is a still further object of the present invention to provide amethod of reducing bone both locally and systemically (whole body),comprising the steps of administering a therapeutic amount of a Wiseagonist to a mammalian patient in need thereof.

Yet another object of the present invention lies in a method ofprotecting a mammalian kidney from chemical injury which results in forexample glomuleronephritis, comprising administering systemically orlocally, to a patient in need thereof, a therapeutic amount of a SOST orWise antagonist.

These objects and others are provided by novel processes utilizingadministration of Sost antagonists or agonists to mammalian patients. Inparticular, Sost antibody antagonists or agonists administered locallywith or without an osteoconductive matrix or in conjunction with anantiresorptive agent. Alternatively, a Sost antibody antagonistsadministered systemically (whole body) in conjunction with anantiresorptive. Desirable Sost antagonists function through LRP5 orLRP6, or comprises an antibody or FAB fragment recognizing any one ofSEQ ID NOS: 1-23.

The above features and advantages are provided by the present inventionwhich utilizes either a Sost or Wise antagonist or a Sost or Wiseagonist to provide bone growth or depletion, respectively.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them unless specifiedotherwise.

As disclosed herein, proteins, particularly antibodies, muteins, nucleicacid aptamers, and peptide that antagonize specific binding of SOST orWISE (Usag-1/ectodin/sostdc1) to their natural receptors may serve as“binding agents” and “SOST antagonists or agonists” or “WISE antagonistsor agonists” of the present invention.

Those of ordinary skill in this art are able to determine theappropriate “therapeutically effective amount” for administering suchagonists and antagonists, as well as methods and schedules for suchadministration

The phrase “specifically (or selectively) binds” or when referring to anantibody interaction, “specifically (or selectively) immunoreactivewith,” refers to a binding reaction between two molecules that is atleast two times the background and more typically more than 10 to 100times background molecular associations under physiological conditions.When using one or more detectable binding agents that are proteins,specific binding is determinative of the presence of the protein, in aheterogeneous population of proteins and other biologics. Thus, underdesignated immunoassay conditions, the specified antibodies bind to aparticular protein sequence, thereby identifying its presence.

Specific binding to an antibody under such conditions requires anantibody that is selected for its specificity for a particular proteinFor example, antibodies raised against a particular protein, polymorphicvariants, alleles, orthologs, and conservatively modified variants, orsplice variants, or portions thereof, can be selected to obtain onlythose polyclonal antibodies that are specifically immunoreactive withSOST, WISE or an LRP, preferably an LRP5 or LRP6 protein and not withother proteins. This selection may be achieved by subtracting outantibodies that cross-react with other molecules. A variety ofimmunoassay formats may be used to select antibodies specificallyimmunoreactive with a particular protein. For example, solid-phase ELISAimmunoassays are routinely used to select antibodies specificallyimmunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, ALaboratory Manual (1988) for a description of immunoassay formats andconditions that can be used to determine specific immunoreactivity).Methods for determining whether two molecules specifically interact aredisclosed herein, and methods of determining binding affinity andspecificity are well known in the art (see, for example, Harlow andLane, Antibodies: A laboratory manual (Cold Spring Harbor LaboratoryPress, 1988); Friefelder, “Physical Biochemistry: Applications tobiochemistry and molecular biology” (W.H. Freeman and Co. 1976)).

Furthermore, Sost or Wise can interfere with the specific binding of areceptor and its ligand by various mechanism, including, for example, bybinding to the ligand binding site, thereby interfering with ligandbinding; by binding to a site other than the ligand binding site of thereceptor, but sterically interfering with ligand binding to thereceptor; by binding the receptor and causing a conformational or otherchange in the receptor, which interferes with binding of the ligand; orby other mechanisms. Similarly, the agent can bind to or otherwiseinteract with the ligand to interfere with its specifically interactingwith the receptor. For purposes of the methods disclosed herein, anunderstanding of the mechanism by which the interference occurs is notrequired and no mechanism of action is proposed. A Wise or Sost bindingagent, such as an anti-Wise or anti-Sost antibody, or antigen bindingfragment thereof, is characterized by having specific binding activity(K_(a)) of at least about 10⁵ M⁻¹, 10⁶ M⁻¹ or greater, preferably 10⁷M⁻¹ or greater, more preferably 10⁸ M⁻¹ or greater, and most preferably10⁹ M⁻¹ or greater. The binding affinity of an antibody can be readilydetermined by one of ordinary skill in the art, for example, byScatchard analysis (Scatchard, Ann. NY Acad. Sci. 51: 660-72, 1949).

The term “antibody” as used herein encompasses naturally occurringantibodies as well as non-naturally occurring antibodies, including, forexample, single chain antibodies, chimeric, bifunctional and humanizedantibodies, as well as antigen-binding fragments thereof, (e.g., Fab′,F(ab′)2, Fab, Fv and rIgG). See also, Pierce Catalog and Handbook,1994-1995 (Pierce Chemical Co., Rockford, Ill.). See also, e.g., Kuby,J., Immunology, 3rd Ed., W.H. Freeman & Co., New York (1998). Suchnon-naturally occurring antibodies can be constructed using solid phasepeptide synthesis, can be produced recombinantly or can be obtained, forexample, by screening combinatorial libraries consisting of variableheavy chains and variable light chains as described by Huse et al.,Science 246:1275-1281 (1989), which is incorporated herein by reference.These and other methods of making, for example, chimeric, humanized,CDR-grafted, single chain, and bifunctional antibodies are well known tothose skilled in the art (Winter and Harris, Immunol. Today 14:243-246(1993); Ward et al., Nature 341:544-546 (1989); Harlow and Lane, supra,1988; Hilyard et al., Protein Engineering: A practical approach (IRLPress 1992); Borrabeck, Antibody Engineering, 2d ed. (Oxford UniversityPress 1995); each of which is incorporated herein by reference).

The term “antibody” includes both polyclonal and monoclonal antibodies.The term also includes genetically engineered forms such as chimericantibodies (e.g., humanized murine antibodies) and heteroconjugateantibodies (e.g., bispecific antibodies). The term also refers torecombinant single chain Fv fragments (scFv). The term antibody alsoincludes bivalent or bispecific molecules, diabodies, triabodies, andtetrabodies. Bivalent and bispecific molecules are described in, e.g.,Kostelny et al., (1992)J Immunol 148:1547, Pack and Pluckthun (1992)Biochemistry 31:1579, Hollinger et al., 1993, supra, Gruber et al.(1994) J Immunol:5368, Zhu et al. (1997) Protein Sci 6:781, Hu et al.(1996) Cancer Res. 56:3055, Adams et al. (1993) Cancer Res. 53:4026, andMcCartney, et al. (1995) Protein Eng. 8:301.

A “humanized antibody” is an immunoglobulin molecule that containsminimal sequence derived from non-human immunoglobulin. Humanizedantibodies include human immunoglobulins (recipient antibody) in whichresidues from a complementary determining region (CDR) of the recipientare replaced by residues from a CDR of a non-human species (donorantibody) such as mouse, rat or rabbit having the desired specificity,affinity and capacity. In some instances, Fv framework residues of thehuman immunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, a humanized antibody will comprise substantiallyall of at least one, and typically two, variable domains, in which allor substantially all of the CDR regions correspond to those of anon-human immunoglobulin and all or substantially all of the framework(FR) regions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin (Jones et al., Nature 321:522-525 (1986); Riechmann etal., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.2:593-596 (1992)). Humanization can be essentially performed followingthe method of Winter and co-workers (Jones et al., Nature 321:522-525(1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al.,Science 239:1534-1536 (1988)), by substituting rodent CDRs or CDRsequences for the corresponding sequences of a human antibody.Accordingly, such humanized antibodies are chimeric antibodies (U.S.Pat. No. 4,816,567), wherein substantially less than an intact humanvariable domain has been substituted by the corresponding sequence froma non-human species.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers, those containing modified residues, and non-naturallyoccurring amino acid polymer.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similarnaturally occurring and non-naturally occurring amino acids are wellknown in the art. Such conservatively modified variants are in additionto and do not exclude polymorphic variants, interspecies homologs, andalleles of the invention. Typically conservative substitutions for oneanother: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamicacid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S),Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g.,Creighton, Proteins (1984)).

“Homologous,” in relation to two or more peptides, refers to two or moresequences or subsequences that have a specified percentage of amino acidresidues that are the same (i.e., about 60% identity, preferably 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, orhigher identity over a specified region, when compared and aligned formaximum correspondence over a comparison window or designated region) asmeasured using a BLAST or BLAST 2.0 sequence comparison algorithms withdefault parameters described below, or by manual alignment and visualinspection (see, e.g., NCBI or the like). The definition also includessequences that have deletions and/or additions, as well as those thathave substitutions, as well as naturally occurring, e.g., polymorphic orallelic variants, and man-made variants. As described below, thepreferred algorithms can account for gaps and the like. Preferably,identity exists over a region that is at least about 25 amino acids inlength, or more preferably over a region that is 50-100 amino acids inlength.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Preferably,default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof one of the number of contiguous positions selected from the groupconsisting typically of from 20 to 600, usually about 50 to about 200,more usually about 100 to about 150 in which a sequence may be comparedto a reference sequence of the same number of contiguous positions afterthe two sequences are optimally aligned. Methods of alignment ofsequences for comparison are well-known in the art. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by thehomology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443(1970), by the search for similarity method of Pearson & Lipman, Proc.Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, 575 Science Dr.,Madison, Wis.), or by manual alignment and visual inspection (see, e.g.,Current Protocols in Molecular Biology (Ausubel et al., eds. 1995supplement)).

Preferred examples of algorithms that are suitable for determiningpercent sequence identity and sequence similarity include the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nuc. AcidsRes. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410(1990). BLAST and BLAST 2.0 are used, with the parameters describedherein, to determine percent sequence identity for the nucleic acids andproteins of the invention. Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation. This algorithm involves first identifying high scoringsequence pairs (HSPs) by identifying short words of length W in thequery sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul et al., supra). These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containingthem. The word hits are extended in both directions along each sequencefor as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, e.g., for nucleotide sequences,the parameters M (reward score for a pair of matching residues;always >0) and N (penalty score for mismatching residues; always <0).For amino acid sequences, a scoring matrix is used to calculate thecumulative score. Extension of the word hits in each direction arehalted when: the cumulative alignment score falls off by the quantity Xfrom its maximum achieved value; the cumulative score goes to zero orbelow, due to the accumulation of one or more negative-scoring residuealignments; or the end of either sequence is reached. The BLASTalgorithm parameters W, T, and X determine the sensitivity and speed ofthe alignment. The BLASTN program (for nucleotide sequences) uses asdefaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=−4 anda comparison of both strands. For amino acid sequences, the BLASTPprogram uses as defaults a wordlength of 3, and expectation (E) of 10,and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl.Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of10, M=5, N=−4, and a comparison of both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a peptide is considered similar to a reference sequence if thesmallest sum probability in a comparison of the test peptide to thereference peptide is less than about 0.2, more preferably less thanabout 0.01, and most preferably less than about 0.001. Log values may belarge negative numbers, e.g., 5, 10, 20, 30, 40, 40, 70, 90, 110, 150,170, etc.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to methods for promoting local bonedeposition in mammals using materials that antagonize Sost proteins.Suitable antagonists may be provided by blocking antibodies. Antibodiesinclude those that specifically bind to any of Sost agonist proteinsaccording to SEQ ID NOS: 1-23 or homologs that are >75% identity andmore preferable antibodies are monoclonal and/or humanized antibodies.It is similarly desirable that the antagonist operates through LRP5 orLRP6. The antagonist may be coadministered or serially administered withan antiresorptive drug if desired to increase or hasten bone formation.For example, the antiresorptive drug may be a bisphosphonate (i.e.fosamax, actonel), a PTH or analog (i.e. Forteo), calcitonin or analog(i.e. Miacalcic), Vitamin D or analog, SERM or analog (i.e. Evista),

These blocking Sost-recognizing antibodies may be made readily by thoseof ordinary skill in this art by conventional techniques. Preferably,these antibodies will be FAB fragments or monoclonal antibodies, andmore preferably, the FAB fragments or monoclonal antibodies will behumanized. Suitable humanized monoclonal antibodies have been created byAmgen, for example. Stowers Institute also provides suitable blockingantibodies designated 4G10, 4B9 and 6E6. Another suitable blockingantibody is 1A12 commercially available from Abcam.

The present invention is directed to methods for reducing bone inmammals using materials that agonize Sost or Wise proteins, byadministering to a mammal a peptide that recognizes any of SEQ ID NOS:1-23. Peptides for treating systemic (whole body) low bone mass diseasesare taught in Applicant's U.S. patent application Ser. No. 11/508,701(U.S. publication No. 20070292444) and in Applicant's US patentapplication publication No. 20040023356. All subject matter of both theSer. Nos. 11/508,701 and 11/613,658 (U.S. publication No. 20070298038)applications and the 20040023356 publication is hereby incorporated byreference.

The present invention is directed to methods for protecting mammaliankidneys from any chemical injury that causes renal damage, for exampleglomuleronephritis, by administering to a mammal a Sost or Wiseantagonist. Such subject matter is disclosed in the Ser. Nos. 11/508,701and 11/613,658 applications and the 20040023356 publication,incorporated by reference.

Other aspects of the present invention are directed towards medicalimplants. Such medical devices and implants include, for example, theosteogenic devices and methods of using the same for repairingendochondral bone and osteochondral defects taught in US patentapplication publication No. 20060177475 to David Rueger et al, publishedAug. 10, 2006, as well as in issued US Patent and published applicationNos. U.S. Pat. No. 6,190,880, 20020169122, 20020187104, 20060252724,20070172479, U.S. Pat. Nos. 5,344,654, 5,324,819, 5,468,845, 6,949,251,6,426,332 and 5,656,593, the subject matter of which is herebyincorporated by reference.

These medical devices generally provide a structural support having animplantable portion preferentially adapted to mechanically engage boneand/or cartilage as taught, for instance, in US patent applicationpublication No. 20060178752 to Joseph Vaccarino III, et al, publishedAug. 10, 2006, the subject matter of which is hereby incorporated byreference. These bone implants desirably comprise an active agent on atleast a portion thereof. As shown by US patent application publicationNo. 20060188542 to John Dennis Bobyn, et al, published Aug. 24, 2006,the subject matter of which is hereby incorporated by reference, theactive agent is preferably formulated to be locally deliverable to boneproximate the implant in sustained-release or in at least a two-phasedrelease scheme. In the latter, a first phase rapidly releases a firstquantity of the active agent, and the second and subsequent phasesgradually release a second quantity of the active agent, whereby boneformation stimulated by the active agent is modulated.

Medical devices such as bone implants feature implantable portionsbearing Sost antagonists foster quicker and more complete bone formationin situ. The implantable portion of the medical device may be desirableat least partially or totally covered or impregnated with a Sostantagonist. It has believed to be helpful to produce the implantableportion of the medical device from a matrix material in which bone canbe formed, to increase permanently retaining the same. This is thoughtto be desirable for materials such as teeth and artificial bone graftsections, and the like. Alternatively, when the implantable sections areload bearing and formed, e.g., of stainless steel, these implantablesections are desirable formed with a Sost antagonist coating. In thatevent, it is desirable to also provide a separate matrix materialconducive to forming new bone growth.

Suitable matrixes include those comprising composite biomaterials havinga sponge-like structure such as those containing, e.g., phosphophorynand/or collagen as taught in Takashi Saito's US patent applicationpublication No. 20060188544, published Aug. 24, 2006, the subject matterof which is hereby incorporated by reference. Such coatings include, forexample, the single and multilayer coatings taught in US patentapplication publication No. 20060204542 to Zongtao Zhang et al,published Sep. 14, 2006, as well as those in U.S. Pat. Nos. 6,949,251,5,298,852, 5,939,039, and 7,189,263 and may be made by conventionalmethods including the methods taught therein, the subject matter ofwhich is hereby incorporated by reference.

Usag-1 (Wise) may be functioning through the Wnt pathway for its role inrenal protection. For this reason and others, a therapeutic amount of aWise antagonist may be administered to a mammalian patient in needthereof so as to protect a kidney from renal damage for exampleglomuleronephritis. In particular, it is especially preferred toadminister such Wise or Sost blocking antibodies so as to protect themammalian kidney from external insult engendered from disease orchemicals, such as toxins or drug therapy.

The present invention also contemplates agents that antagonize bindingof SOST and/or WISE to its native receptor(s) (“SOST antagonist”). SOSTantagonist include a peptidomimetic, which is an organic molecule thatmimics the structure of a peptide; or a peptoid such as a vinylogouspeptoid.

The present invention also contemplates agents that agonize binding ofSOST and/or WISE to its native receptor(s) (“SOST agonists). SOST orWise agonists include a peptidomimetic, which is an organic moleculethat mimics the structure of a peptide; or a peptoid such as avinylogous peptoid.

Preferred embodiments of the present invention include SOST antagoniststhat are preferably SOST antibodies, WISE antibodies or LRP antibodies,although the invention also contemplates inhibitory peptides and smallmolecular inhibitors as described above. Antibodies of the invention arepreferably chimeric, more preferably humanized antibodies, ideallymonoclonal antibodies preferably raised against murine proteins, mostpreferably murine SOST.

SOST, WISE, or LRP antagonist antibodies, including anti-SOSTantibodies, may be raised using as an immunogen, such as a substantiallypurified full length protein, such as murine SOST, but may also be aSOST, WISE or LRP protein of human, mouse or other mammalian origin. Theimmunogen may be prepared from natural sources or producedrecombinantly, or a peptide portion of a protein, which can include aportion of the cystiene knot domain, for example, a synthetic peptide. Anon-immunogenic peptide may be made immunogenic by coupling the haptento a carrier molecule such bovine serum albumin (BSA) or keyhole limpethemocyanin (KLH), or by expressing the peptide portion as a fusionprotein. Various other carrier molecules and methods for coupling ahapten to a carrier molecule are well known in the art and described,for example, by Harlow and Lane (supra, 1988).

Particularly useful antibodies for performing methods of the inventionare monoclonal antibodies that that specifically bind to LRP molecules,WISE or, most preferably, SOST. Such antibodies are particularly usefulwhere they bind SOST with at least an order of magnitude greateraffinity than they bind another protein. Methods for creating chimericantibodies, including humanized antibodies, is discussed in greaterdetail below.

1. Production of Recombinant Antibody

Methods for producing both monoclonal and polyclonal antibodies fromidentified proteins or peptides are well known in the art. In order toprepare recombinant chimeric and humanized antibodies that may functionas SOST antagonists of the present invention, the nucleic acid encodingnon-human antibodies must first be isolated. This is typically done byimmunizing an animal, for example a mouse, with prepared Sost or Wise oran antigenic peptide derived therefrom. Typically mice are immunizedtwice intraperitoneally with approximately 50 micrograms of proteinantibody per mouse. Sera from immunized mice can be tested for antibodyactivity by immunohistology or immunocytology on any host systemexpressing such polypeptide and by ELISA with the expressed polypeptide.For immunohistology, active antibodies of the present invention can beidentified using a biotinconjugated anti-mouse immunoglobulin followedby avidin-peroxidase and a chromogenic peroxidase substrate.Preparations of such reagents are commercially available; for example,from Zymad Corp., San Francisco, Calif. Mice whose sera containdetectable active antibodies according to the invention can besacrificed three days later and their spleens removed for fusion andhybridoma production. Positive supernatants of such hybridomas can beidentified using the assays common to those of skill in the art, forexample, Western blot analysis.

The nucleic acids encoding the desired antibody chains can then beisolated by, for example, using hybridoma mRNA or splenic mRNA as atemplate for PCR amplification of the heavy and light chain genes [Huse,et al., Science 246:1276 (1989)]. Nucleic acids for producing bothantibodies and intrabodies can be derived from murine monoclonalhybridomas using this technique [Richardson J. H., et al., Proc NatlAcad Sci USA 92:3137-3141 (1995); Biocca S., et al., Biochem and BiophysRes Comm, 197:422-427 (1993) Mhashilkar, A. M., et al., EMBO J14:1542-1551 (1995)]. These hybridomas rovide a reliable source ofwell-characterized reagents for the construction of antibodies and areparticularly useful once their epitope reactivity and affinity has beencharacterized. Isolation of nucleic acids from isolated cells isdiscussed further in Clackson, T., et al., Nature 352:624-628 (1991)(spleen) and Portolano, S., et al., supra; Barbas, C. F., et al., supra;Marks, J. D., et al., supra; Barbas, C. F., et al., Proc Natl Acad SciUSA 88:7978-7982 (1991) (human peripheral blood lymphocytes). Humanizedantibodies optimally include at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin [Jones etal., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329(1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].

A number of methods have been described to produce recombinantantibodies, both chimeric and humanized. Controlled rearrangement ofantibody domains joined through protein disulfide bonds to form chimericantibodies may be utilized (Konieczny et al., Haematologia, 14(1):95-99,1981). Recombinant DNA technology can also be used to construct genefusions between DNA sequences encoding mouse antibody variable light andheavy chain domains and human antibody light and heavy chain constantdomains (Morrison et al., Proc. Natl. Acad. Sci. USA, 81(21):6851-6855,1984.).

DNA sequences encoding the antigen binding portions or complementaritydetermining regions (CDR's) of murine monoclonal antibodies may begrafted by molecular means into the DNA sequences encoding theframeworks of human antibody heavy and light chains (Jones et al.,Nature, 321(6069):522-525, 1986; Riechmann et al., Nature,332(6162):323-327, 1988.). The expressed recombinant products are called“reshaped” or humanized antibodies, and comprise the framework of ahuman antibody light or heavy chain and the antigen recognitionportions, CDR's, of a murine monoclonal antibody.

Other methods for producing humanized antibodies are described in U.S.Pat. Nos. 5,693,762; 5,693,761; 5,585,089; 5,639,641; 5,565,332;5,733,743; 5,750,078; 5,502,167; 5,705,154; 5,770,403; 5,698,417;5,693,493; 5,558,864; 4,935,496; 4,816,567; and 5,530,101, eachincorporated herein by reference in their entirety.

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778, which is incorporated by reference) can be adaptedto produce single chain humanized antibodies to Sost or Wise.

2. Purification of Recombinant Antibody

Affinity purification of an antibody pool or sera provides apractitioner with a more uniform reagent. Methods for enriching antibodygranulation inhibitors using antibody affinity matrices to form anaffinity column are well known in the art and available commercially(AntibodyShop, c/o Statens Serum Institut, Artillerivej 5, Bldg. P2,DK-2300 Copenhagen S). Briefly, an antibody affinity matrix is attachedto an affinity support (see e.g.; CNBR Sepharose (R), PharmaciaBiotech). A mixture comprising antibodies is then passed over theaffinity matrix, to which the antibodies bind. Bound antibodies arereleased by techniques common to those familiar with the art, yielding aconcentrated antibody pool. The enriched antibody pool can then be usedfor further immunological studies, some of which are described herein byway of example.

Another approach uses recombinant bacteriophage to produce largelibraries. Using the “phage method” (Scott and Smith, Science249:386-390, 1990; Cwirla, et al, Proc. Natl. Acad. Sci., 87:6378-6382,1990; Devlin et al., Science, 49:404-406, 1990), very large librariescan be constructed (106-108 chemical entities). A second approach usesprimarily chemical methods, of which the Geysen method (Geysen et al.,Molecular Immunology 23:709-715, 1986; Geysen et al. J. ImmunologicMethod 102:259-274, 1987; and the method of Fodor et al. (Science251:767-773, 1991) are examples Furka et al. (14th InternationalCongress of Biochemistry, Volume #5, Abstract FR:013, 1988; Furka, Int.J. Peptide Protein Res. 37:487-493, 1991), Houghton (U.S. Pat. No.4,631,211, issued December 1986) and Rutter et al. (U.S. Pat. No.5,010,175, issued Apr. 23, 1991) describe methods to produce a mixtureof peptides that can be tested as agonists or antagonists.

3. Identification of Sost Antagonists

The present invention provides methods for identifying diagnostic andtherapeutic SOST antagonists. Several exemplary methods for identifyingsuch antagonists are described herein, including cell-based and in vitrotechniques. A general method of identifying SOST antagonists involvesevaluating the effects of antagonist candidates on bone deposition undercontrolled conditions. Preferably bone deposition is determined usingmicro-CT techniques on live animals. Preferred animals include rodents,more preferred are primates. Femur and vertebrae bones are particularlyuseful subjects for such study.

Briefly, the test animal is treated with a predetermined dose of a SOSTantagonist candidate. A control animal is treated with a controlsolution, preferably a non-irritating buffer solution or other carrier.

It also has been found that successful implantation of the osteogenicfactors for endochondral bone formation requires association of theproteins with a suitable carrier material capable of maintaining theproteins at an in vivo site of application. The carrier should bebiocompatible, in vivo biodegradable and porous enough to allow cellinfiltration.

The proteins of this invention, including fragments thereof, also may beused to raise monoclonal or polyclonal antibodies capable of bindingspecifically to an epitope of Sost, Wise, or LRP. These antibodies maybe used, for example, in Sost or Wise antagonists or agonistspurification protocols.

The Sost or Wise antagonists or agonists are useful in clinicalapplications in conjunction with a suitable delivery or support system(matrix). As disclosed herein, the matrix may be combined with Sost orWise antagonists or agonists to induce endochondral bone formationreliably and reproducibly in a mammalian body. The matrix is made up ofparticles of porous materials. The pores must be of a dimension topermit progenitor cell migration into the matrix and subsequentdifferentiation and proliferation. The particle size should be withinthe range of 70 um-850 um, preferably 70 um-420 um, most preferably 150um-420 um. It may be fabricated by close packing particulate materialinto a shape spanning the bone defect, or by otherwise structuring asdesired a material that is biocompatible, and preferably biodegradablein vivo to serve as a “temporary scaffold” and substratum forrecruitment of migratory progenitor cells, and as a base for theirsubsequent anchoring and proliferation. Useful matrix materialscomprise, for example, collagen; homopolymers or copolymers of glycolicacid, lactic acid, and butyric acid, including derivatives thereof; andceramics, such as hydroxyapatite, tricalcium phosphate and other calciumphosphates. Combinations of these matrix materials also may be useful.

When the SOST antagonist candidate is delivered in a carrier, thecontrol solution is ideally the carrier absent the SOST antagonistcandidate. Multiple doses of the SOST antagonist candidate may beapplied to the test animal, preferably following a predeterminedschedule of dosing. The dosing schedule may be over a period of days,more preferably over a period of weeks.

Once the dosing schedule has been completed, both test and controlanimals are examined to determine the level of bone deposition present.This may be accomplished by any suitable method, but is preferablyperformed on live animals using x-ray equipment. Methods for micro-CTexamination of bones in animals are well known in the art. A SOSTantagonist candidate suitable for use as a SOST antagonist is identifiedby noting significant bone deposition in the test animal when comparedto the control animal. Ideally bone deposition in the test bone(s) ofthe test animal should be at least 10%, more preferably 20%, mostpreferably 30% or 40% or more bone deposition than is present in thesame bones of the control animal. Where necessary, levels of bonedeposition may be calculated by determining the volume of bonedeposition present in each animal. Calculations may be performed byconstructing a 3-dimensional image of the bone deposition andcalculating the volume from the image with the aid of e.g.,histomorphometry.

In an exemplary embodiment, localized injection in situ of a SOSTantagonist candidate, for example a monoclonal antibody describedherein, may be made into a test animal, with a control animal receivingan equal volume of control solution without the SOST antagonistcandidate. Identical dosing should be done on a weekly basis for fourweeks. Suitable dosage will depend on the nature of the particular SOSTantagonist candidate being tested. By way of example, in dosing itshould be noted that systemic injection, either intravenously,subcutaneously or intramuscularly, may also be used. For systemicinjection of a SOST antagonist candidate or a SOST antagonist oragonist, dosage should be about 5 mg/kg, preferably more preferablyabout 15 mg/kg, advantageously about 50 mg/kg, more advantageously about100 mg/kg, acceptably about 200 mg/kg. dosing performed by nebulizedinhalation, eye drops, or oral ingestion should be at an amountsufficient to produce blood levels of the SOST antagonist candidatesimilar to those reached using systemic injection. The amount of SOSTantagonist candidate that must be delivered by nebulized inhalation, eyedrops, or oral ingestion to attain these levels is dependent upon thenature of the inhibitor sed and can be determined by routineexperimentation. It is expected that, for systemic injection of themonoclonal antibody SOST antagonist candidates described herein,therapeutic levels of the antibody may be detected in the blood one weekafter delivery of a 15 mg/kg dose.

SOST antagonists may also be identified using a process known ascomputer, or molecular modeling, which allows visualization of thethree-dimensional atomic structure of a selected molecule and therational design of new compounds that will interact with the molecule.The three-dimensional construct typically depends on data from x-raycrystallographic analyses or NMR imaging of the selected molecule. Themolecular dynamics require force field data. The computer graphicssystems enable prediction of how a new compound will link to the targetmolecule and allow experimental manipulation of the structures of thecompound and target molecule to perfect binding specificity. redictionof what the molecule-compound interaction will be when small changes aremade in one or both requires molecular mechanics software andcomputationally intensive computers, usually coupled with user-friendly,menu-driven interfaces between the molecular design program and theuser.

An example of the molecular modelling system described generally aboveconsists of the CHARMm and QUANTA programs, Polygen Corporation,Waltham, Mass. CHARMm performs the energy minimization and moleculardynamics functions. QUANTA performs the construction, graphic modellingand analysis of molecular structure. QUANTA allows interactiveconstruction, modification, visualization, and analysis of the behaviorof molecules with each other.

A number of articles review computer modeling of drugs interactive withspecific proteins, such as Rotivinen, et. al., Acta PharmaceuticaFennica 97, 159-166 (1988); Ripka, New Scientist 54-57 (Jun. 16, 1988);McKinaly and Rossmann, Annu. Rev. Pharmacol. Toxiciol. 29, 111-122(1989); Perry and Davies, OSAR: Ouantitative Structure-ActivityRelationships in Drug Design pp. 189-193 (Alan R. Liss, Inc. 1989);Lewis and Dean, Proc. R. Soc. Lond. 236, 25-140 and 141-162 (1989); and,with respect to a model receptor for nucleic acid components, Askew, etal., J. Am. Chem. Soc. 111, 1082-1090 (1989). Askew et al. constructed anew molecular shape which permitted both hydrogen bonding and aromaticstacking forces to act simultaneously. Askew et al. used Kemp's triacid(Kemp et al., J. Org. Chem. 46:5140-5143 (1981)) in which a U-shaped(diaxial) relationship exists between any two carboxyl functions.Conversion of the triacid to the imide acid chloride gave an acylatingagent that could be attached via amide or ester linkages to practicallyany available aromatic surface. The resulting structure featured anaromatic plane that could be roughly parallel to that of the atoms inthe imide function; hydrogen bonding and stacking forces converged fromperpendicular directions to provide a microenvironment complimentary toadenine derivatives.

Other computer programs that screen and graphically depict chemicals areavailable from companies such as BioDesign, Inc., Pasadena, Calif.,Allelix, Inc, Mississauga, Ontario, Canada, and Hypercube, Inc.,Cambridge, Ontario. Although these are primarily designed forapplication to drugs specific to particular proteins, they can beadapted to design of drugs specific to regions of RNA, once that regionis identified.

4. Screening Compound Libraries

Whether identified from existing SOST antagonists or from molecularmodelling techniques, SOST antagonists generally must be modifiedfurther to enhance their therapeutic usefulness. This is typically doneby creating large libraries of compounds related to the SOST antagonist,or compounds synthesized randomly, based around a core structure. Inorder to efficiently screen large and/or diverse libraries of SOSTantagonist candidates, a high throughput screening method is necessaryto at least decrease the number of candidate compounds to be screenedusing the assays described above. High throughput screening methodsinvolve providing a combinatorial chemical or peptide library containinga large number of potential therapeutic compounds (potential modulatoror ligand compounds). Such “combinatorial chemical libraries” or“candidate libraries” are then screened in one or more assays, asdescribed below, to identify those library members (particular chemicalspecies or subclasses) that are able to promote bone deposition. Thecompounds thus identified can serve as conventional “lead compounds” orcan themselves be used as potential or actual therapeutics.

Accordingly, the present invention provides methods for high throughputscreening of SOST antagonists candidates. The initial steps of thesemethods allow for the efficient and rapid identification ofcombinatorial library members that have a high probability of being SOSTantagonists. These initial steps take advantage of the observation thatSOST antagonists are also LRP or SOST binding agents. Any method thatdetermines the ability of a member of the library, termed a bindingcandidate, to specifically bind to SOST, WISE or an LRP protein issuitable for this initial high throughput screening. For example,competitive and non-competitive ELISA-type assays known to one ofordinary skill in the art may be utilized.

Binding candidates that are found to bind SOST, WISE or an LRP proteinwith acceptable specificity, e.g., with a K_(a) for SOST, WISE or an LRPprotein of at least about 10⁵ M⁻¹, 10⁶ M⁻¹ or greater, preferably 10⁷M⁻¹ or greater, more preferably 10⁸ M⁻¹ or greater, and most preferably10⁹ M⁻¹ or greater, are SOST antagonist candidates and are screenedfurther, as described above, to determine their ability to promote bonedeposition.

5. Therapeutic Applications

Individuals to be treated using methods of the present invention may beany mammal, for example local increase in bone may be used for fracturehealing, fusion (arthrodesis), orthopedic reconstruction, andperiodontal repair. Systemic increase in bone would be for treatment oflow bone mass, i.e. osteoporosis. Bone reduction would be used to treatunwanted heterotopic bone formation, ossification of longitudinalligament, ossification during cervical stenosis, or osteosarcoma. Suchindividuals include a dog, cat, horse, cow, or goat, particularly acommercially important animal or a domesticated animal, moreparticularly a human.

In therapeutic use SOST antagonists generally will be in the form of apharmaceutical composition containing the antagonist and apharmaceutically acceptable carrier. Pharmaceutically acceptablecarriers are well known in the art and include aqueous solutions such asphysiologically buffered saline or other buffers or solvents or vehiclessuch as glycols, glycerol, oils such as olive oil or injectable organicesters. The selection of a pharmaceutically acceptable carrier willdepend, in part, on the chemical nature of the SOST antagonist, forexample, whether the SOST antagonist is an antibody, a peptide or anonpeptide.

A pharmaceutically acceptable carrier may include physiologicallyacceptable compounds that act, for example, to stabilize the SOSTantagonist or increase its absorption, or other excipients as desired.Physiologically acceptable compounds include, for example,carbohydrates, such as glucose, sucrose or dextrans, antioxidants, suchas ascorbic acid or glutathione, chelating agents, low molecular weightproteins or other stabilizers or excipients. One skilled in the artwould know that the choice of a pharmaceutically acceptable carrier,including a physiologically acceptable compound, depends, for example,on the route of administration of the SOST antagonist and on itsparticular physio-chemical characteristics.

Generally, such carriers should be nontoxic to recipients at the dosagesand concentrations employed. Ordinarily, the preparation of suchcompositions entails combining the therapeutic agent with buffers,antioxidants such as ascorbic acid, low molecular weight (less thanabout 10 residues) polypeptides, proteins, amino acids, carbohydratesincluding glucose, maltose, sucrose or dextrins, chelating agents suchas EDTA, glutathione and other stabilizers and excipients. Neutralbuffered saline or saline mixed with nonspecific serum albumin areexemplary appropriate diluents.

The pharmaceutical compositions of the present invention may be preparedfor administration by a variety of different routes. In general, thetype of carrier is selected based on the mode of administration.Pharmaceutical compositions may be formulated for any appropriate mannerof administration, including, for example, topical, oral, nasal,intrathecal, rectal, vaginal, sublingual or parenteral administration,including subcutaneous, intravenous, intramuscular, intrasternal,intracavernous, intrameatal, or intraurethral injection or infusion. Apharmaceutical composition (e.g., for oral administration or delivery byinjection) may be in the form of a liquid (e.g., an elixir, syrup,solution, emulsion or suspension). A liquid pharmaceutical compositionmay include, for example, one or more of the following: sterile diluentssuch as water for injection, saline solution, preferably physiologicalsaline, Ringer's solution, isotonic sodium chloride, fixed oils that mayserve as the solvent or suspending medium, polyethylene glycols,glycerin, propylene glycol or other solvents; antibacterial agents;antioxidants; chelating agents; buffers such as acetates, citrates orphosphates and agents for the adjustment of tonicity such as sodiumchloride or dextrose. A parenteral preparation can be enclosed inampoules, disposable syringes or multiple dose vials made of glass orplastic. The use of physiological saline is preferred, and an injectablepharmaceutical composition is preferably sterile.

The methods of the present invention include application of SOSTantagonists in cocktails including other medicaments, for example,antibiotics, fungicides, and anti-inflammatory agents. Alternatively,the methods may comprise sequential dosing of an afflicted individualwith a SOST antagonist and one or more additional medicaments tooptimize a treatment regime. In such optimized regimes, the medicaments,including the granulation inhibitor may be applied in any sequence andin any combination.

The SOST, Wise, or LRP antagonists or agonists of the present inventionmay also be included in slow release formulations for prolongedtreatment following a single dose. In one embodiment, the formulation isprepared in the form of microspheres. The microspheres may be preparedas a homogenous matrix of a SOST antagonist with a biodegradablecontrolled release material, with optional additional medicaments as thetreatment requires. The microspheres are preferably prepared in sizessuitable for infiltration and/or injection, and injected systemically,or directly at the site of treatment.

The formulations of the invention are also suitable for administrationin all body spaces/cavities, including but not limited to pleura,peritoneum, cranium, mediastinum, pericardium, bursae or bursal,epidural, intrathecal, intraocular, intra-articular, intra-discal,intra-medullary, perispinal, etc.

Some slow release embodiments include polymeric substances that arebiodegradable and/or dissolve slowly. Such polymeric substances includepolyvinylpyrrolidone, low- and medium-molecular-weight hydroxypropylcellulose and hydroxypropyl methylcellulose, cross-linked sodiumcarboxymethylcellulose, carboxymethyl starch, potassiummethacrylatedivinylbenzene copolymer, polyvinyl alcohols, starches,starch derivatives, microcrystalline cellulose, ethylcellulose,methylcellulose, and cellulose derivatives, β-cyclodextrin, poly(methylvinyl ethers/maleic anhydride), glucans, scierozlucans, mannans,xanthans, alzinic acid and derivatives thereof, dextrin derivatives,glyceryl monostearate, semisynthetic glycerides, glycerylpalmitostearate, glyceryl behenate, polyvinylpyrrolidone, gelatine,agnesium stearate, stearic acid, sodium stearate, talc, sodium benzoate,boric acid, and colloidal silica.

Slow release agents of the invention may also include adjuvants such asstarch, pregelled starch, calcium phosphate mannitol, lactose,saccharose, glucose, sorbitol, microcrystalline cellulose, gelatin,polyvinylpyrrolidone. methylcellulose, starch solution, ethylcellulose,arabic gum, tragacanth gum, magnesium stearate, stearic acid, colloidalsilica, glyceryl monostearate, hydrogenated castor oil, waxes, andmono-, bi-, and trisubstituted glycerides. Slow release agents may alsobe prepared as generally described in WO94/06416.

The amount of SOST, Wise, or LRP antagonist or agonists administered toan individual will depend, in part, on the disease and/or extent ofinjury. Methods for determining an effective amount of an agent toadminister for a diagnostic or a therapeutic procedure are well known inthe art and include phase I, phase II and phase III clinical trials.Generally, an agent antagonist is administered in a dose of about 0.01to 200 mg/kg body weight when administered systemically, and at aconcentration of approximately 0.1-100 μM when administered directly toa wound site. The total amount of SOST antagonist or agonists can beadministered to a subject as a single dose, either as a bolus or byinfusion over a relatively short period of time, or can be administeredusing a fractionated treatment protocol, in which the multiple doses areadministered over a more prolonged period of time. One skilled in theart would know that the concentration of a particular SOST antagonistrequired to provide an effective amount to a region or regions of injurydepends on many factors including the age and general health of thesubject as well as the route of administration, the number of treatmentsto be administered, and the nature of the SOST antagonist, includingwhether the SOST antagonist is an antibody, a peptide, or a nonpeptidemolecule. In view of these factors, the skilled artisan would adjust theparticular dose so as to obtain an effective amount for efficaciouslypromoting bone deposition for therapeutic purposes.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for clarity and understanding, it willbe readily apparent to one of ordinary skill in the art in light of theteachings of this invention that certain changes and modifications maybe made thereto without departing from the spirit and scope of theappended claims.

What I claim is:
 1. A method for promoting bone growth in a humansubject being treated with a humanized sclerostin-recognizing antibody,comprising administering an antiresorptive drug to the subject.
 2. Themethod of claim 1, wherein the subject has previously been treated withthe antiresorptive drug.
 3. The method of claim 1, wherein the subjectis being treated with the humanized sclerostin-recognizing antibodysubcutaneously.
 4. The method of claim 1, wherein the antiresorptivedrug is a bisphosphonate, calcitonin, Vitamin D, or selective estrogenreceptor modulator (SERM).
 5. The method of claim 1, wherein theantiresorptive drug is Vitamin D.
 6. The method of claim 1, wherein theantibody is formulated in a pharmaceutical composition.
 7. The method ofclaim 6, wherein the pharmaceutical composition comprises apharmaceutically acceptable carrier selected from the group consistingof: water, saline, buffers, solvents, vehicles, oils, polyethyleneglycols, glycerin, propylene glycol, carbohydrates, antioxidants,chelating agents, low molecular weight proteins, proteins, amino acids,stabilizers, excipients, and antibacterial agents.
 8. The method ofclaim 7, wherein the buffer comprises acetate, the carbohydratecomprises sucrose, and the pharmaceutically acceptable carrier comprisesa polyethylene glycol.
 9. The method of claim 1, wherein the subject isbeing treated with the humanized sclerostin-recognizing antibodysubcutaneously, the antibody is a sclerostin blocking antibody, and theantiresorptive drug is Vitamin D.
 10. The method of claim 1, wherein theadministration of the antiresorptive drug results in increased boneformation relative to bone formation prior to administration of thehumanized sclerostin-recognizing antibody and the antiresorptive drug.11. The method of claim 1, wherein the administration of theantiresorptive drug results in increased bone formation relative to ahuman subject not being treated with the humanizedsclerostin-recognizing antibody.
 12. The method of claim 1, wherein theantiresorptive drug comprises at least one of bisphosphonate,calcitonin, Vitamin D, or SERM.
 13. The method of claim 12, wherein theantiresorptive drug is Vitamin D.
 14. The method of claim 1, wherein theantiresorptive drug is serially administered to the human subject beingtreated.
 15. A method for increasing bone density in a human subjectwith low bone mass being treated with a sclerostin-recognizing antibody,comprising serially administering an antiresorptive drug to the subject.16. The method of claim 15, wherein the sclerostin is human sclerostinhaving the sequence of SEQ ID NO:22.
 17. The method of claim 15, whereinthe sclerostin-recognizing antibody is humanized.
 18. The method ofclaim 15, wherein the human subject has at least one of osteoporosis andlow bone density.
 19. The method of claim 15, wherein the antiresorptivedrug comprises at least one of bisphosphonate, calcitonin, Vitamin D, orSERM.
 20. The method of claim 15, wherein the antiresorptive drug isVitamin D.
 21. The method of claim 15, wherein the human subject haspreviously been treated with an antiresorptive drug.
 22. The method ofclaim 15, wherein the human subject has not previously been treated withan antiresorptive drug.
 23. The method of claim 15, wherein theantiresorptive drug is administered alone.
 24. The method of claim 15,wherein the antiresorptive drug is administered alone and following thehuman subject with low bone mass being treated with thesclerostin-recognizing antibody.